Proteinaceous particles of the same order of magnitude as virions, which have affinity for particular cell types are potentially useful as delivery vehicles for bringing a desired therapeutic molecule carried by the particles into contact with cells of that type. The therapeutic molecule may be, for example, an antigen capable of stimulating a target cell of the immune system, or nucleic acid for incorporation within the target cell, the latter objective being a minimum requirement for successful gene therapy applications.
The use of recombinant retroviruses as vehicles in gene therapy is gaining increasing support. Natural retroviruses comprise a GAG protein "core" or "capsid", these terms being used interchangeably, the capsid being enveloped by the envelope proteins (strictly these are glycoproteins but will be referred to herein simply as envelope proteins), and within the capsid is packaged the viral genomic RNA. By application of recombinant nucleic acid technology, the structure of the capsid and envelope proteins, and of the genomic RNA, of a natural retrovirus may be altered. Thus, recombinant particles comprising capsid and envelope proteins can be used to carry foreign nucleic acid, for insertion into cells which are members of the host range defined by the receptor binding specificity of the envelope proteins. A major goal in retroviral gene therapy is to alter the host range of recombinant retroviral particles to target the particles to particular cell types and to provide efficient transduction. The preferred virus for developing retroviral gene therapy is generally accepted to be murine leukemia virus (MLV).
Outside the specific area of gene therapy, it would also be desirable to adapt proteinaceous particles for targeting to specific cell types, for example to deliver specific antigens to target cells of the immune system by presenting such antigens on proteinaceous virion-like particles whose envelope proteins carry sequences having an affinity for the desired target cell type.
Specific interactions have been proposed between the transmembrane (TM) domain of envelope (ENV) proteins and the matrix (MA) domain of GAG protein for selective incorporation of the envelope proteins into the self-assembling particle. In addition, the cell receptor binding domain of retroviral envelope proteins has been located near the N-terminus. In MLV, two discontinuous receptor recognition regions termed VRA and VRB have been identified (Battini et al. (1992) J. Virol. 66:1468-1475)). Any modification of the envelope protein to confer specific cell targeting properties on an enveloped capsid must retain the regions important for incorporation of the envelope, yet possess an altered receptor binding domain for targeted infection.
The use of targeted retroviral particles as carriers has been attempted before. Young et al. (1990) attempted to selectively incorporate CD4 into ALV particles by fusing CD4 to a truncated viral envelope protein. Curiously, there was incorporation of wild-type CD4 but very poor incorporation of the fusion protein. The absolute efficiency of incorporation was difficult to determine, and it is possible that both wild-type and fusion CD4 were poorly picked up by the virus.
In WO 94106920, retrovirus particles displaying a functional antibody fragment is disclosed. These inventors fused the gene coding for a functional antibody fragment (250 amino acids) at the 5'-end of the gene coding for the complete Moloney Murine Leukemia Virus envelope protein (Pr80env) and following transfection in an ecotropic packaging cell line recovered particles with specific hapten binding properties. That such modified viruses can infect cells using antibody-hapten recognition has not yet been demonstrated.
In WO 94/11524 the receptor binding region of the envelope protein of murine leukemia viruses were replaced by the mature rabbit alpha-1 acid glycoprotein antigen binding site region of an antibody.
In the foregoing publications,.wherein the envelope proteins have undergone gross changes, problems arose such as incorrect processing and transport of modified envelope proteins, low-level infectivity, poor envelope incorporation on the particle, or low viral titers on expression. It therefore appears that in order to maintain the envelope functions, other than cell targeting, i.e. correct processing and transport to the cell surface, incorporation onto the capsid particle, infection of the targeted cell and good expression yields, there should be minimal disruption to the envelope protein.
Valsesia-Wittmann et al. (J.Virol 68: 4609; 1994) used the 16 amino acid FLA-16 RGD peptide inserted into the strictly avitropic RSV envelope at specific sites to redirect tropism. Some insertion sites destroyed the ability of the envelope to be processed or incorporated in viral particles, and the most successful insertion site enabled the mutant virus produced to infect mammalian cells, but only at very low titres, namely 10.sup.2 /ml, or 10.sup.3 /ml when the virus was deglycosylated.